HCV infects around 3% of worldwide population (Williams R. Hepatology 2006; 44: 521-526). Most of the infected individuals evolve to chronicity (Amoroso P, y cols., J Hepatol 1998; 28: 939-944). Hepatitis C infection is one of the principal causes of chronic hepatic damage, cirrhosis, liver failure and liver cancer (Hoofnagle J H. Hepatology 2002; 36 (5 Suppl 1): S21-S29). Currently, HCV infection is the principal cause of liver transplantation in first world countries. Additionally, HCV infection has been related with extra-hepatic manifestations as type II cryoglobulinemia, membranoproliferative glomerulonephritis, porphyria cutanea tarda, among others. At present, a preventive vaccine against this virus is not available and the conventional antiviral treatments in use, based on the combination of pegylated interferon (IFN)+ribavirin, are effective in less than 50% of cases. Furthermore, the aforementioned treatments cause multiples adverse events (Ghany M G y cols., Hepatology. 2009; 49 (4):1335-74).
HCV belongs to the Hepacivirus genus of the Flaviviridae family. It is an enveloped virus, which viral particles are around 50 and 70 nm in diameter and are associated to very low density lipoproteins (VLDL) (Popescu C I y Dubuisson J. Biol Cell. 2009; 102 (1):63-74). The viral genome is a positive stranded ribonucleic acid (RNA) of approximately 9.6 kb. The genome encodes for a viral polyprotein that is processed co- and post-translationally in at least 10 viral proteins: Core, E1, E2, p7 (structural proteins) and nonstructural proteins: NS2, NS3, NS4A, NS4B, NS5A, NS5B (Bartenschlager R y Lohmann V. 2000. J Gen Virol. 81: 1631-48).
There are several important obstacles to the development of an effective vaccine against HCV. This pathogen is an RNA virus that can rapidly mutate adapting to the host environment. This contributes to the high diversity of the multiple viral isolates identified worldwide. Six major HCV genotypes have been identified, which can differ up to 30% in nucleotide sequence (Simmonds P. J Hepatol. 1999; 31 Suppl 1:54-60). The greatest heterogeneity is observed in the hypervariable region of HCV E2 protein, where an epitope potentially targeted by neutralizing antibodies is found. In fact, HCV circulates in the body as a heterogeneous population of viral molecules, this phenomenon is known as quasiespecies (Simmonds P. J Gen Virol. 2004; 85 (Pt 11):3173-88). It has been demonstrated that mutations constitute a way of viral escape to specific humoral and cellular immune response developed by the host.
It should be stressed that HCV causes persistent infection in immunocompetent individuals, despite the occurrence of an active immune response (Lechmann et al., Semin Liver Dis 2000, 20, 211-226). Currently, several viral effects that contribute to the persistence of the infection, by encouraging irrelevant immune responses and preventing an effective immune response, have been elucidated. These effects are detected over both innate and acquired immunity (Grakoui A y cols. Science 2003, 302 (5645): 659-62). There are evidences supporting that an ineffective immune response against HCV, not only fails to eliminate this pathogen, but it also contributes to liver damage.
So far, the immunological parameters that correlate with protection and clarification against HCV have not been completely defined. However, the induction of potent and sustained cellular immune response against different HCV antigens is considered particularly relevant (Lechmann et al., Semin Liver Dis 2000, 20, 211-226). In HCV chronically infected patients the impairment of the specific T lymphocyte response is particularly significant. Several mechanisms seem to contribute to this effect; one of them is the action of regulatory T cells.
Almost all immunization strategies have been attempted to develop a vaccine against HCV. Some of those strategies include: recombinant proteins, synthetic peptides, virus like particles, naked deoxyribonucleic acid (DNA) and recombinant viruses. All viral antigens have been evaluated as targets in vaccine candidates against HCV. Most of the vaccine candidates are at the stage of immunogenicity studies in animal models. Nevertheless, at present some candidates have reached clinical evaluation, they have demonstrated to be safe and immunogenic, but a clear clinical impact has not been demonstrated yet (Alvarez-Lajonchere L, Dueñas-Carrera S. Int Rev Immunol. 2012; 31(3):223-42).
The development of a subunit protein vaccine candidate was one of the first strategies evaluated to obtain an HCV vaccine. Some of those candidates based on structural antigens have achieved limited protection against viral challenges in animal models. Such is the case of chimpanzees immunized with an E1 and E2 oligomer. Seven chimpanzees were immunized, five of them became protected and two became infected, but then cleared the virus without reaching chronicity (Choo y cols., Proc Natl Acad Sci USA 1994, 91, 1294-1298). This protection correlated with the presence of antibodies able to inhibit the interaction between E2 protein and human cells (Rosa y cols., Proc Natl Acad Sci USA 1996, 93, 1759-1763).
A recombinant E1 protein, from a 1 b genotype isolate, was purified as homodimers (Maertens y cols., Acta Gastroenterol Belg 2000, 63, 203). Two chimpanzees chronically infected with HCV received 9 doses of 50 μg of this recombinant E1 protein. Vaccination improved liver histology, cleared viral antigens from the liver and reduced alanine aminotransferase levels. However, serum RNA levels did not change during treatment and hepatic inflammation and viral antigens reappeared after treatment conclusion. An association between high anti-E1 antibodies levels and liver damage reduction was observed (Maertens et al., Acta Gastroenterol Belg 2000, 63, 203). An E1 protein variant formulated in alum was evaluated in humans. This candidate was safe and immunogenic, inducing specific antibodies and linfoproliferative responses (Nevens F, y col., Hepatology. 2003; 38 (5):1289-96). However, the administration of this candidate did not affect the clinical course of HCV infection, as patients did not clear the virus, and no liver histological improvement was observed. The protein subunits approach, as a disadvantage, has not induced a strong cellular immune response in some cases. This approach may have another drawback: the insertion of regions involved in the different mechanisms of HCV-specific immune response impairment induced by the pathogen at different levels (Grakoui A y cols., Science 2003, 302 (5645): 659-62).
Two vaccine candidates based on mixtures of synthetic peptides, including T lymphocytes epitopes, have also reached clinical trials (Yutani y cols., Cancer Sci 2009.100(10): 1935-42, Klade y cols., Gastroenterology 2008.134(5): 1385-95). Both candidates induced specific immune responses and had low reactogenicity in HCV chronically infected patients during Phase I and II clinical trials conducted so far (Alvarez-Lajonchere L, Dueñas-Carrera S. Int Rev Immunol. 2012; 31 (3):223-42). Nevertheless, these vaccine candidates have not shown significant effect on viral load or have had a transient effect. Taking into account that these candidates have not induced any improvement over liver histology, their clinical impact is still to be demonstrated. Different epitopes for CD4+ and CD8+ T cells that might be important for viral clearance have been identified throughout HCV polyprotein. These findings support the synthetic peptides based vaccine strategy. Different peptides including Core, NS4 and NS5 epitopes, alone or with lipids moieties, have induced strong T cytotoxic responses in mice (Shirai et al., J Infect Dis 1996, 173, 24-31; Hiranuma et al., J Gen Virol 1999, 80, 187-193; Oseroff et al., Vaccine 1998, 16, 823-833). The principal disadvantage of this approach is that those peptides without T helper function may be poor immunogens. In addition, the efficacy of a vaccine depends frequently on the induction of a multivalent and broad spectrum immune response against several antigens. As the number of peptides included in a vaccine increases, the formulation complexity rises from all viewpoints. These limitations are weaknesses of this approach.
On the other hand, different recombinant viral vectors have been evaluated as vaccine candidates for HCV. Defective recombinant adenoviruses are attractive candidates, due to their liver tropism, their capacity to induce humoral and cellular immune responses and the possibility to be administered by oral and parenteral routes. Recombinant adenoviruses expressing HCV structural proteins induce antibodies responses against each of these proteins (Makimura et al., Vaccine 1996, 14, 28-36). Besides, after mice immunization with Core and E1 recombinant adenovirus, a specific T cytotoxic immune response is detected against these antigens (Bruna-Romero et al., Hepatology 1997, 25, 470-477). Although these are encouraging results; some problems related to the use of recombinant adenoviruses in gene therapy have raised doubts about their safety in humans. At present, a vaccine candidate based on HCV recombinant adenovirus is being evaluated in clinical trials with good results on immunogenicity, but without evidence of clinical impact (Barnes y cols., Sci Transl Med. 2012; 4(115): 115). The use of others recombinant viral vectors, such as vaccinia, fowlpox and canarypox containing different HCV genes have induced strong T cytotoxic and helper responses in mice (Shirai et al., J Virol 1994, 68, 3334-3342; Large et al., J Immunol 1999, 162, 931-938). Particularly, a modified vaccinia virus Ankara, recombinant for HCV nonstructural antigens NS3-NS5, has been evaluated in clinical trials in humans (Fournillier y cols., Vaccine. 2007; 25 (42):7339-53). This candidate was immunogenic and well tolerated in a phase I clinical trial in HCV chronically infected patients. Similar to the peptide approach, the effect over de viral load was transient and observed only in a fraction of the vaccinees; therefore, the clinical impact is still to be demonstrated. In general, the vaccine candidates based on recombinant viruses are hampered by safety and regulatory issues related with their application. DNA immunization has been extensively studied as a strategy for HCV vaccine development. Studies in animal models have showed the capacity of these candidates to induce cellular and humoral immune responses against almost all HCV antigens (Alvarez-Lajonchere L, Dueñas-Carrera S, Hum Vaccin. 2009; 5 (8):568-71). Two vaccine candidates that include DNA immunization plasmids containing sequences encoding HCV antigens are in clinical trials in humans (Alvarez-Lajonchere L, Dueñas-Carrera S, Int Rev Immunol. 2012; 31 (3):223-42). In one case, it is a DNA vaccine expressing NS3 to NS5 proteins, administered by electroporation (Sällberg M, y cols., Expert Opin Biol Ther. 2009; 9 (7):805-15). In the other case, it is a vaccine composition based on the mixture of a recombinant core protein and a DNA plasmid that expresses HCV structural antigens (Castellanos M, y cols., J Gene Med. 2010; 12 (1):107-16). Both candidates have demonstrated to be safe, well tolerated and have induced specific immune responses in immunized subjects (Alvarez-Lajonchere L, Dueñas-Carrera S, Int Rev Immunol. 2012; 31 (3):223-42). In neither of these two cases, the effect over HCV infection course or a sustained histological improvement, have been demonstrated. DNA vaccines, despite their potential advantages related to their simplicity and stability, face important regulatory challenges. Their principal limitation seems to be related to their insufficient immunogenicity in humans, phenomenon not completely understood so far, and that differs considerably with the results obtained in animal models.
According to the aforementioned elements, the development of a prophylactic or therapeutic vaccine against HCV is an unsolved problem. The present invention is directed precisely towards this goal.